Homologous recombination occurs when a broken or damaged chromosome uses a homologous chromosome as repair template. Recombination underpins DNA replication and genome stability, and is essential for chromosome segregation during meiosis. Our long-term goal is to understand the mechanism and regulation of recombination. The fundamental recombination reaction is the formation of Joint Molecule (JM) intermediates via pairing and strand-exchange between a broken chromosome and a homologous template. A multiplicity of DNA nucleases and helicases function during every step of recombination and assigning in vivo functions to specific enzymes, and understanding how they interact during recombination remain challenging issues. This proposal will investigate the in vivo roles of nucleases and helicases in JM metabolism. Specific Aim 1. To Characterize the Pathways of Holliday Junction Resolution. We have identified five activities responsible for essentially all JM resolution and crossing-over during meiosis. These include mismatch repair factors, Exo1, Mlh1 and Mlh3; XPF-family nuclease, Mus81-Mms4; Slx4, which forms two distinct nuclease complexes; the recently identified HJ resolvase, Yen1; and RecQ helicase, Sgs1, which functions in a complex to dissociate dHJs rather than resolve them. Molecular and genetic approaches will be used to assign specific roles to these activities in vivo and test specific models of meiotic JM resolution. An assays system has been developed that, for the first time, allows detection of JMs formed in vivo during mitotic DSB-repair. This unique tool will be used to determine the roles of JM resolving factors in mitotic DSB-repair. Specific Aim 2. To Determine the Roles of DNA Helicases in Regulating Joint Molecule Metabolism. Three DNA helicases, Sgs1, Srs2 and Mph1 appear to function independently to suppress crossing-over during mitotic DSB-repair. However, during meiosis, these anti-crossover factors must be inhibited at sites of crossing-over, but may be required to complete non-crossover recombination. Molecular assays will be used to determine the roles of these helicases in regulating JM formation during meiosis and during mitotic DSB-repair. A specific model of meiotic JM formation and the roles of helicases in this process will be examined. We will also test the idea that Srs2 is prevented from disrupting filaments of RecA proteins, Rad51 and Dmc1, during meiosis by the so-called mediator proteins. Finally, we will examine the molecular roles of two additional helicases: Hrq1, a recently identified fungal homolog of RecQ4, which is mutated in Rothmund-Thomson syndrome; and the helicase/nuclease Dna2, which is defective for crossing-over during mitotic DSB-repair Defective recombination is associated with infertility, pregnancy miscarriage and genetic disease. In somatic cells is especially relevant for cancer. An understanding of the molecular processes of homologous recombination will help us better understand the etiology of these disorders. PUBLIC HEALTH RELEVANCE: Homologous recombination is required for sexual reproduction and chromosome repair. Defects in this process are linked to human infertility, miscarriage and genetic diseases, especially cancer. A greater understanding of the mechanism and regulation of homologous recombination will help us better understand the etiology of these diseases and design novel therapies.